Autotrophic Nitrifying Bacteria and Their Practical Application in a Freshwater Aquarium

Aquarium Bacteria and Filtration Manifesto

By: Kmuda

Bacteria play a significant role inside our tanks. Without it, we would not be able to maintain sufficient water quality to possess healthy fish. For the purpose of this article, we will discuss the two primary types of bacteria that affect water quality. Autotrophic Bacteria, which are the nitrifiers, and Heterotrophic Bacteria, which are the sludge reducers (consumers of organics) and the denitrifiers. It is not the intent of this article to discuss tank cycling, but the information it contains can be useful in understanding this process. The intent is to provide information allowing the creation of an environment able to sustain healthy and stable colonies of beneficial bacteria.

Beneficial bacteria should be treated as if they were another fish in your tank. Fortunately, ensuring a stable and healthy population of these bacteria requires basically the same environment your fish require. Armed with a little bit of knowledge, you can guarantee the health and viability of your fish by ensuring the health and viability of these bacteria.

Diagram of the Nitrogen Cycle

Autotrophic Nitrifying Bacteria.

These are the “good guys”. Autotrophic bacteria (autotrophs) are so named because they possess the ability to make their own food. Some species of autotrophs accomplish this via photosynthesis (using sunlight, oxygen, and water), but the autotrophs that perform a vital role in our aquariums make their own food via chemosytnesis (using oxygen and ammonia or nitrite, both of which are nitrogenous waste), which is why they are called nitrifiers (or nitrogen-fixing bacteria). They convert ammonia that naturally occurs in our tanks into nitrite, they then break down the nitrite into nitrate (a process known as the “Nitrogen Cycle”.)

Species

There is much debate within science (not to mention the hobby) as to exactly which species of autotrophic bacteria perform the vital functions of the “Nitrogen Cycle” in our tanks. Historically, it was thought the species associated with the conversion of ammonia into nitrite was Nitrosomonas europaea and that the species associated with the conversion of nitrite into nitrate were Nitrobacter, Nitrospina, and/or Nitrococcus. This “belief” was based upon the correct fact that these are the bacteria primarily responsible for maintaining the nitrogen cycle in soil. To this day, most bacterial additives (quick cycle products) available to aquarium hobbyist contain these bacteria. Unfortunately, it has been discovered that this is not the correct combination of bacteria that exists in our tanks. It’s no wonder that aquarist worldwide have come to the combined conclusion that these “quick cycle” products do not work. They (as in, the industry) are packaging bacteria that function in soil and selling it to us as something that works in water. It doesn’t.

Research conducted by Timothy A. Hovanec (formerly of Marineland Labs, now the founder of Dr. Tim’s Aquatics) has identified the correct combination of autotrophic bacteria performing the nitrogen cycle in our tanks are Nitrosomonas marina (converts ammonia to nitrite) and Nitrospira (converts nitrite to nitrate). I am aware of only two “bacterial additive” products that contain these bacteria, Dr. Tim’s Aquatics “One and Only” and Tetra SafeStart. United Pet Group has purchased Marineland. In the process, Dr. Tim bought out Marineland Labs (the creator of BioSpira, which is no longer available), forming “Dr. Tim’s Aquatics”. United Pet Group also owns Tetra. Tetra has subsequently released Tetra Safe-Start, which appears to be BioSpira repackaged in a form that does not require constant refrigeration (just as One and Only does not require refrigeration). It may be that availability of actual functioning product, containing the correct combination of living bacteria, may remain limited to these two products due to patent restrictions. Dr. Tim and the former Marineland Labs (now Dr. Tim’s Aquatics) hold the patent and United Pet Group/Tetra received co-ownership via their purchase of Marineland.

Autotrophic Nitrifying Bacteria Pre-Requisite: Ammonia

Regardless of the specific species (or packaged products), two things are required in order for autotrophic bacteria to exist (and function) in our tanks, a source of ammonia and plenty of oxygen. The population of autotrophic bacteria is entirely dependent upon the amount of ammonia being produced. Small amounts of ammonia will result in small colonies of bacteria. Large amounts of ammonia will result in large colonies of bacteria, provided sufficient surface area is available for the bacteria to colonize. To state the obvious, this means that you cannot drop an Oscar into a tank formerly containing a few guppies and not expect a cycle (a toxic ammonia spike followed by a toxic nitrite spike). The population of autotrophic bacteria will only be sized to support the ammonia production of the guppies, not the mass bio-overload of an Oscar. Should you make such a mistake, since autotrophic nitrifying bacteria only divide once every 12-20 hours, it can take a significant period of time for the bacteria population to catch up to the amount of ammonia being produced. From scratch, it can take up to 8 weeks for these bacteria to fully colonize a tank in sufficient quantity to eliminate ammonia and nitrite. Another issue is that nitrospira (the bacteria that converts nitrite into nitrate) cannot multiply in water that contains significant concentrations of ammonia. There is even evidence suggesting that existing populations of nitrospira become dormant when ammonia is present in high concentrations. So if you are cycling a tank, the nitrospira bacteria will not even begin to colonize until the Nitrosomonas marina bacteria population is sufficient in size to bring ammonia concentrations under control. If you experience an ammonia spike in an established tank, you can expect a subsequent nitrite spike, with the nitrite spike being a “double-whammy” as the nitrite oxidizing bacteria may have to wake up from dormancy, then increase in population to account for the abnormally high nitrite levels.

Each bite of food you feed to your fish contributes to ammonia production. Fish consume the food, the food is broken down and, as with all vertebrates, their kidneys remove the resulting toxins. However, unlike us, freshwater fish do not release these toxins as urea/ammonia in their urine. Ammonia is removed from their body, via the gills, in the form of ammonium. While the primary ammonia source in an aquarium is fish respiration, uneaten food in the substrate, “gunk” build-up in the filters, fish poo, decaying plants, dead bacteria, as well as other forms of organic matter, are consumed by heterotrophic bacteria (and fungi), and converted into ammonia. In an established tank, colonies of of bacteria grow in direct relation to the amount of ammonia being produced by these various factors, maintaining undetectable levels of ammonia (and nitrite) provided their basic needs are met and there is not a sudden increase in the amount of ammonia being produced. As an example, ammonia (and subsequent nitrite) spikes can result from over feeding. If you accidentally dump an excess of flake food into a tank and don’t clean it up, or a fish dies and you don’t remove the corpse, you can expect an ammonia/nitrite spike. The colony of bacteria that exists in the tank is sized to support the ammonia production of the fish population. The extra ammonia resulting from the decaying food or fish corpse would be an additional bioload that the colony of autotrophic nitrifying bacteria is not sized to deal with. The same scenario plays out if you add new fish to a tank. If that fish adds a substantial bioload onto to the tank (with bioload being limited for the purpose of this discussion to ammonia production), it can take a while for the colonies of autotrophic bacteria to increase in size to deal with the additional ammonia being produced.

You can minimize these concerns by providing optimal oxygen levels, an excess of surface area for bacteria to colonize, an excess flow rate to bring the ammonia/nitrite laden water into contact with the nitrifying bacteria, and an optimal temperature conducive to autotrophic nitrifying bacteria growth and efficiency. pH also plays a significant role, but manipulating the pH carries more risk than reward and a less than ideal pH (ideal for bacteria) can be compensated for by temperature, available oxygen, flow rate, and surface area.

Autotrophic Nitrifying Bacteria Pre-Requisite: Oxygen

Secondly, as mentioned, for autotrophic bacteria to exist, in addition to ammonia, there must be oxygen (the more the better). The minimum concentration of dissolved oxygen required to support effective colonies of autotrophic bacteria is 2ppm. Oxygen in my tanks is maintained at 9ppm. Any method that increases oxygen concentrations for our fish will result in available oxygen for autotrophic nitrifying bacteria. Air stones, bubble curtains, powerheads, filter outflows, etc… all result in water turbulence and agitation of the water surface, which pulls oxygen from the atmosphere into the water column. (On an unrelated side note, the same action also pulls in whatever else is in the atmosphere -cigarette smoke, air freshener, perfume, household chemicals, dirty sock smells, etc…). However, unlike our fish, the autotrophic bacteria supporting the nitrogen cycle only have to be wet, they do not have to be submerged. This is where Wet/Dry filtration, which includes Biowheels, establishes its advantage over submerged biomedia. Media that is exposed to the atmosphere is able to utilize oxygen from the atmosphere. This eliminates any concern of insufficient oxygen to support the bacteria, it removes the bacteria from competition with the fish for oxygen, which results in more oxygen being available to the fish, and it eliminates the possibility of heterotrophic bacteria out competing the autotrophic bacteria for oxygen.

It is important to understand that autotrophic nitrifying bacteria compete with heterotrophic bacteria, as well as our fish, for oxygen. And while autotrophs require higher oxygen concentrations than the heterotrophs, heterotrophic bacteria are the most prolific and capable consumer of oxygen in our tanks, even more so than our fish (more on this later).

Autotrophic bacteria do not actively seek out ammonia and nitrite. These compounds must be brought to them, which is why filtration flow rates and absence of filter bypass are of such importance. Enough water must be cycled through filter media in sufficient a time frame to prevent ammonia and nitrite concentrations from building up, standing in line (so to speak) to come into contact with the autotrophic nitrifying bacteria inhabiting the filter media. Autotrophic nitrifying bacteria do not exist in any measurable quantity in the water column itself. These bacteria exist (assuming it’s an established tank and the autotrophs are not being out competed by heterotrophs) on every surface in the aquarium, including on the gravel/sand, on decorations, on plants, on those silly skeleton/treasure chest ornaments, on the walls of the tank, on the filter tubes, and especially, in the filter media. The more surface area that is available, the larger the colony of autotrophic nitrifying bacteria that can exist (and the better it is able to grow), which is why “surface area” is such an important consideration when purchasing bio-media. It’s also why “bypass” is a significant concern when selecting a filter. Water that flows around filter media is not coming into contact with the autotrophic nitrifying bacteria, so the ammonia/nitrite that water contains will still exist as the water exits the filter. As this relates to Biowheel filters, flow rate is more important than surface area. Since there is no bypass with water coming into contact with a bio-wheel, and it exists in an oxygen rich environment, which allow the bacteria to be more efficient, it requires substantially less surface area than standard submerged filtration media. It is also feasible that water flowing over a biowheel becomes so saturated with oxygen that it degasses CO2, artifically (temporarily) inflating the pH, making the bacteria even more efficient.

Just as autotrophic bacteria compete with heterotrophic bacteria for oxygen, they also compete with heterotrophic bacteria for space. As with all living things, they (both types of bacteria) die. When they die they leave behind their corpse, and the corpse of these dead bacteria pile up on top of each other. The end result, they (along with other ditrius) fill in the pores of our filter media, reducing the available surface area. The benefit of these “pores” cannot be over rated. With them, a single Biomax type ceramic ring will possess a surface area equivalent to 1” of gravel in a 10-gallon tank. Without them, the same ring contains no more surface area than a single 1” rock in a 10-gallon tank. Dead bacteria are also an additional food source for living heterotrophic bacteria, increasing their populations. When heterotrophic bacteria consume the dead bacteria, the end result is the same as if they were consuming fish poo, with that result being ammonia (then nitrite, then nitrate). This process is also conducive to an environment more favorable to heterotrophic bacteria than autotrophic, which we do not want (not here anyway, more on that later). For these reasons, it is important that our biomedia is regularly cleaned. I take things to an extreme in this regard. Once per month, biomedia is removed from my canisters and rinsed well in tank water. I usually dump the media in a bucket of tank water and then stir the media around by hand. In addition, I will remove one tray worth of biomedia, replacing it with either new media or recycled media. The removed media is taken to the sink, rinsed well, and then boiled. Once clean, it is dried for several days on a towel and then placed into a zip lock bag for future reuse (when rotating out another tray of media.) For media that may be exceptionally dirty, I’ve even taken it a step further by soaking the media overnight in a solution containing APPlus+ Plastic Plant Cleaner before boiling it, making sure it is well rinsed before I boil it. I find that this returns ceramic media rings, such as Hagen and Fluval BioMax, to “like new” condition. When using APPlus+ Plastic Plant Cleaner to clean ceramic biomedia, I use it at the same strength identified on the bottle for cleaning plastic plants. Below is a photo of approximately 3 year old Hagen Biomax from one of my filters following this cleaning process (using APPlus+ Plastic Plant Cleaner). You can't tell a difference between it and brand new product.

Biowheels do not require such extensive cleaning, as they do not rely upon surface area in the same fashion as standard media. I do regularly shake out my bio-wheels in a bucket of tank water (every month or so), but I’ve never gone beyond that. If a wheel gets so dirty that it stops spinning, and you cannot sufficiently clean it in a bucket of water to get it spinning again, it should probably be replaced.

Biowheel Disclaimer

It should be mentioned, since I have identified an obvious affinity for Biowheels, that Biowheels also have shortcomings. Lower cost models, such as the Penguin line, that depend on water flowing out of the filter (via gravity) to spin the wheel, experience an issue with partial bypass. Upper end models, such as the Emperor line, utilize spray bars to spray water over the wheel, ensuring there is no bypass as long as the wheel is spinning. Which is the remaining “shortcoming”, Biowheels have a habit of stopping. While I have rarely experienced this issue myself, it is common, especially in locations with hard water as calcium deposits build up on the wheel making them too heavy to spin. The few times one of my wheels has stopped spinning it was because of operator error (I placed the filter lid on wrong or pushed the aquarium lid back to tight against the filter casing, placing pressure on the Biowheel, and it stopped spinning). That said, I have Biowheels that have been in constant use for over a decade, some as long as 18 years, and they continue to spin.

Autotrophic Nitrifying Bacteria and pH

pH has a significant effect on autotrophic nitrifying bacteria. At a pH below 6.5, they become inhibited and cease being efficient in oxidizing ammonia and nitrite. Maximum rates of nitrification occur at pH values above 7.2, with optimal nitrification occurring around 8.3. At a pH of 8.3, nitrifying bacteria operate at near 100% effectiveness. At a pH of 7.0, their efficiency drops to less than 50%. At a pH of 6.5, it drops to 30%, and at a pH of 6.0, it drops to 10% optimal efficiency.

At pH values below 6.0, autotrophic nitrifying bacteria don’t die, they just stop functioning and reproducing. We are fortunate that at these low pH values, most ammonia (NH3) is ionized into its non-toxic form, ammonium (NH4).

What does this mean in a practical sense? For starters, if cycling a tank (fishless cycling, of course) it would be of benefit to artificially inflate the pH of the water to between 8.0 and 8.3. Baking Soda is an inexpensive and effective method of accomplishing this. At this higher pH, nitrifying bacteria are more efficient and are able to reproduce faster than at a lower pH, which should decrease the cycling time. This is not a recommendation for an established tank, only when performing a fishless cycle.

It also ties back into flow rates and available surface space. At a pH of 8.3, it would require approximately ½ the quantity of bacteria to oxidize a specific amount of ammonia and nitrite as it would at a pH of 7.0. This is because at a pH of 7.0 the autotrophic nitrifying bacteria are only operating at about 50% efficiency. You compensate for this by increasing the colony size of autotrophic nitrifying bacteria. Twice as much bacteria operating at 50% efficiency can oxidize the same amount of ammonia as ½ the quantity of bacteria operating at near 100% efficiency. We, as aquarist, enable the increased colony of bacteria by providing ample surface area and flow rates.

Old Tank Syndrome:

These same concepts also apply to what is known as “Old Tank Syndrome” (OTS). OTS occurs because of a process known as bio-acidification. Autotrophic nitrifying bacteria, in the act of oxidizing ammonia and nitrite, release acids. These acids erode away the pH buffer (carbonate hardness, KH). If frequent enough water changes are performed, the pH buffer is replenished, the pH remains stable, and the bacteria remain functional. If, however, insufficient water changes are performed, eventually the pH buffer will be overwhelmed and the pH will drop dramatically. Once the pH falls below 6.0, autotrophic denitrifying bacteria cease to function and reproduce. However, because the pH is so low, the ammonia is ionized into its non-toxic form, and the fish continue on as if everything was OK. If an unassuming aquarist then performs a water change, the pH will increase significantly. Ammonia is converted back into its toxic form and fish start to die. In addition to the ammonia being converted back into its toxic form, the autotrophic nitrifying bacteria have to awaken from (what is the equivalence of) suspended animation. This takes time and ammonia is still being produced in the tank by the fish. In the meantime, the autotrophic nitrifying bacteria may have a difficult time increasing their colony size to account for the increased ammonia levels, as available surface area may be limited. While the autotrophic bacteria were dormant, the heterotrophic bacteria continued to grow, especially considering the environment we are discussing is the result of insufficient maintenance so the levels of organics will be significantly higher than they should be. With this extensive food source, heterotrophic bacteria will have a field day, potentially inhabiting all available surface area, leaving the autotrophs no place to colonize.

You prevent this scenario by frequent water changes, by performing tank maintenance, including filter media maintenance discussed previously, and by providing an over abundance of surface area (which also involves filter media maintenance). Should you ever encounter this scenario, an important component to recovery is properly (safely, in tank water) cleaning the existing media and, if possible, adding more.

Autotrophic Nitrifying Bacteria and Temperature

The single biggest factor affecting the growth of autotrophic nitrifying bacteria is temperature. Optimum growth is achieved between 77-86F. Growth rate declines to 50% at a temp of 64F and there will be no growth at a temp of 39F. At temperatures exceeding 95 degrees the bacteria start to experience life threatening stress, likely due to enzyme disruption. The bacteria will die at temps below 32F and above 120F.

Nitrite oxidizing bacteria are more susceptible to temperature extremes than are the ammonia oxidizing bacteria.

Again, the question should be asked, what does this mean in a practical sense? It suggests that, if the fish are compatible, the tank temperature should be maintained somewhere between 77F and 80F. Almost all tropical fish are perfectly happy within this temperature range and you are providing the optimal conditions for the bacteria that keep the fish alive. It also identifies that if cycling a tank, increasing the temp to around 82F (in conjunction with increasing the pH) should provide optimal conditions for autotrophic bacteria growth, reducing cycling time. But beware, this only applies to a “fishless cycle”. You should not increase the temperature (or pH) if cycling with fish, unless you are prepared for the fish to die. Ammonia toxicity is directly related to temperature and pH in that the higher the temperature, or pH, the more toxic is the ammonia.

Autotrophic Nitrifying Bacteria Pre-Requisite: Water

The species of autotrophic bacteria responsible for maintaining the nitrogen cycle in our tanks require water. If they are allowed to dry out, they die. Allowing them to dry out does not cause them to go into suspended animation, nor do they form viable spores. It results in their absolute death. This means that if you allow your filter media and/or gravel to dry out, or if your bio-wheel stops spinning and you don’t discover it, all of the beneficial nitrifying bacteria that has colonized those surfaces will die and subsequent colonies of autotrophic bacteria, once the media and/or gravel is re-submerged in water, will have to start from a baseline of zero (the beginning).

Autotrophic Nitrifying Bacteria and Chlorine/Chloramines

Autotrophic nitrifying bacteria are highly susceptible to chlorine or chloramines, with the nitrite oxidizers being the most fragile. Chlorine and Chloramines are added to water supplies to kill bacteria, including autotrophic nitrifying bacteria. Filters should be turned off when refilling the tanks from a water change and dechlorinator should be added before the refill starts. Several hours following water changes, you should be testing to ensure that neither ammonia nor nitrite concentrations are increasing. Ammonia oxidizing bacteria stand a better chance of surviving a mistake (for example, not using a dechlorinator). Therefore, ammonia tests may not reveal an issue when a nitrite test would. At the same time, if the ammonia oxidizing bacteria gets nuked (from medication or chlorine dosage), then ammonia will not be converted to nitrite, so a nitrite test will not indicate an issue where the ammonia test would. It is essential you test for both.

Autotrophic Nitrifying Bacteria and Medications

Like chlorine, antibacterial and many anti-parasitic medications are indiscriminate killers of bacteria, good and bad. If you dose a tank with antibacterial medications, chances are that in addition to the bad bacteria, the good bacteria will be killed as well. Again, the nitrite oxidizing bacteria are more fragile than the ammonia oxidizing bacteria. So, following use of medications, it is important that you test for both ammonia and nitrite, not just one of the two.

When antibacterial medications are required, use of a hospital tank is preferred. By using a hospital tank, you can dose with medications without risking the autotrophic nitrifying bacteria in your main tank. I maintain sponge filters in each of my tanks. These filters are easily transferred over to a hospital tank and are moved at the same time as the fish to be medicated. Moving these filters does not impact the main tank yet they provide continued filtration in the hospital tank, unless (until) the medication kills off the beneficial bacteria they contain.

If I am dealing with an issue that requires the entirety of the main tank to be medicated, I move one of my canister filters over to the hospital tank. The autotrophic nitrifying bacteria this filter contains is supported by feeding the hospital tank with pure ammonia (same process as a “fishless cycle”.) This is another reason why two filters are always a good ideal, providing flexibility. When the medication process is completed, the filter is moved back to the main tank (after emptying out the water it contains). If the main tank is in a “cycle”, you should mix the bio-media between the unaffected filter (the one that was moved to the hospital tank) and the filter that remained on the main tank. This usually allows full recovery from a “mini-cycle” within a short amount of time (as in overnight to 24 hours). If you have multiple tanks, you can “steal” media from a healthy tank to place into another. However, you should never move media from an infected tank to an uninfected one without first sterilizing it. Boiling the media for at least 30 minutes will ensure that any bacteria or parasites the media contained are killed.

A point of concern when discussing this process is that if you are treating for a bacterial ailment and that bacteria exists within the filter media, you run the risk of reintroducing the bacteria once you move the filter from the hospital tank back to the main tank. This is a risk you have to weigh for yourself and I would judge the risk on each individual case. In most instances, the “cure” associated with use of antibacterial medications is actually that the medications kicked back the bacteria enough that the fish is able to develop immunity from further infection. In which case, there is no harm in moving a filter from the main tank to a hospital tank, artificially supporting that filter via the fishless cycle method, then moving this filter back to the main tank once treatment is completed. However, there are some bacterial infections that require absolute sterilization. If all of the fish in the main tank do not survive treatment, then tanks, filters, and associated media should be sterilized, including the hospital tank to which the filter was temporarily moved.

I would consider the risk of re-infection higher if dealing with a parasitic infection. However, most parasites cannot exist for long without a fish host. Again, use of this process has to be judged on a case-by-case bases. If the parasite you are treating for cannot exist without a fish host, then there is no danger, provided you leave the filter on the hospital tank long enough for the “intermediary” stage of the parasite to die off.

This process ties back to issues discussed previously. If you have multiple filters, an excess of surface area (via filtration media), along with an excess of flow rate, combined with sufficient oxygen, and optimal temperature, you can remove a filter from a tank without causing major ammonia/nitrite spikes, as the colonies of beneficial bacteria can easily expand. At worst, those spikes will be very short-lived. On the other hand, if you are under filtered, or filtered “just enough”, you have limited options.

Autotrophic Nitrifying Bacteria and Power Outages.

Extra steps should be taken to help our beneficial bacteria survive power outages. In the event of an extended power outage, if you intend to run your filters off of a generator, then you’re set. The rest of us must be a bit more creative.

A vital component of any aquarist’s emergency kit should be battery-powered air pumps. A three-pack of the Azoo brand can be purchased from Drs. Foster and Smith for around $10. So there is really no excuse for not having them on hand. In the event of an extended power outage, my emergency kit contains these pumps, plus bubble disks. The bubble disks are placed on the aquarium floor, on top of the substrate, and connected to the battery powered air pumps. Trays of biomedia are then removed from the canister filters and stacked on top of the bubble disk. This allows enough water flow through the trays to maintain populations of bacteria (as well as provide continued bio-filtration). Bio-wheels are taken out and floated in the tank.

The one thing you do not want is to leave the media in your filters, especially in canister filters. In an extended power outage, the oxygen content of the water inside the canisters will expire resulting in the beneficial bacteria shutting down and entering hibernation. In addition, many species of heterotrophic bacteria can continue to function in an absence of oxygen. Since these bacteria can multiply quickly, they can easily over run the autotrophic nitrifying bacteria we are trying to preserve. It is entirely possible, depending on the amount of organics contained within the filters, that these heterotrophic bacteria will cause putrification, which will result in the death of the autotrophic nitrifying bacteria within the canister. If nothing else, remove the media from the canisters and place it into the main tank, where the autotrophic nitrifying bacteria stand a better chance of survival.